Booij TH, Klop MJ, Yan K, Szántai-Kis C, Szokol B, Orfi L, van de Water B, Keri G,

Price LS.


3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting.


Identification of selective c-Met/EGFR inhibitors in a Vichem EGFR/c-Met inhibiting compound library.

A compound screen was performed where 80 Vichem compounds are tested at 10, 3.16 and 1µM. Compounds were divided over triplicate plates and co-exposed with either HGF (20ng/mL) or EGF (20ng/mL). PCA was trained on unstimulated and stimulated controls separately for plates exposed to HGF or EGF, respectively, to obtain a principal component that could separate c-Met and EGFR responses. Data points represent mean determinations from 3 wells. Controls are color-coded as indicated in the legend. For stimulated (top middle) and unstimulated controls (bottom left), a 2D-density estimation (contour lines) is shown.